Dear Premkumar,

Apart from gel electrophoresis, you can also run internal control genes such as GAPDH / beta actin  if RNA samples from human / animal, 16S rDNA amplification for bacteria and etc... to ensure the cDNA successfully converted.

Thanks

I'll rather amplify it with specific primers that should not work on genomic DNA: design two primers separated by a large intron, and then use a small amount of your diluted cDNA (ie: if you retrotranscribed 500ng, in 20µl, use 5µl of a 1:10 dilution).

Don't waste precious material in electrophoresis.

I think easiest way is to just to a simple PCR/qPCR with internal control such as GAPDH or ACTB. just make sure the primers target CDS. If the agarose gel show distinct band of the housekeeping gene meaning your cDNA is fine

If you want a sensitive method to assess cDNA product size/integrity, you can label the cDNA during the RT step. Traditionally this is done using 32P dCTP, but can also be done using biotin-11-dUTP. Make a dNTP mix with a 1:1:0.6:0.4 ratio of dGTP:dCTP:dATP:dTTP: biotin-11-dUTP. Separate a sample of the cDNA reaction product on agarose, blot onto nylon and detect with streptavidin-AP or streptavidin-HRP.  If you have biotinylated DNA standards, you could quantify cDNA as well by dot-blot.

RT-PCR analysis for a known intron-containing gene using a pair of intron spanning  prmers.

Dear Premkumar Ganesan,

I strongly suggest that you use primers from an housekeppinge gene. Those primers should amplify different lenght fragment in gDNA and cDNA.

That way you will confirm that:

-Your cDNA samples are free of gDNA

-Your cDNA samples were synthesized properly

-The synthesis efficiency was roughly the same for all samples (if the PCR amplification band has the same intensity for all samples)

Best of luck

To be sure you produce cDNA from transcripts and follow the genomic DNA contamination the solution was already given above, PCR for a selected gene using  primers around an intron and analyse the band length in an agarose gel.

I just do Q-PCR for GAPDH actin etc reference control.

HI, You must determine goodness of cDNA synthesis using primers of reference genes. Like 18s or 16s rRNA or actin.

Best, A.

I would suggest directly go for your RT-PCR rather than checking it on gel. as on gel it will give you a smear so better to confirm through PCR. Hope it will be helpful.